Structural rearrangement of native and processed pea starches following simulated digestion in vitro and fermentation characteristics of their resistant starch residues using human fecal inoculum.

Pea starches, in both native (NPS) and retrograded-autoclaved forms (RAPS), were subjected to simulated gastrointestinal (GI) digestion in vitro, their multi-scale structural characteristics, morphological features, molecular distribution and thermal properties were characterized. A gradual increase in the short-/long-range crystallinity, melting enthalpy of gelatinization on increasing digestion time was observed for both the native and retrograded-autoclaved pea starch samples based on the X-ray diffraction, Fourier-transform infrared spectra, solid-state 13CNMR and differential scanning calorimetry measurements. It was especially noticed that the growth rate of crystallinity and double helices, as well as the decrease in Mw values were evidently greater for RAPS than for NPS. To investigate how different molecular fine structure of pea starch substrate affects the gut microbiota shifts and dynamic short-chain fatty acid profile, their resistant starch residues obtained from both native and retrograded-autoclaved pea starch after 8 h of simulated GI tract digestion was used as the fermentation substrate. The levels of acetate, propionate and butyrate gradually increased with the increasing fermentation time for NPS and RAPS. In comparison to the blank control (i.e., the group without the addition of carbohydrate), the fermented NPS and RAPS obviously resulted in an increased abundance of Firmicutes and Bacteroidetes, accompanied by a decrease in Proteobacteria, Actinobacteria and Verrucomicrobia. Both NPS and RAPS promoted different shifts in the microbial community at the genus level, with an increase in the abundance of Bacteroides, Megamonas and Bifidobacterium, as well as a reduction in the abundance of Fusobacterium, Faecalibacterium and Lachnoclostridium in comparison to the blank control samples.

Evaluation and Optimization of Sample Handling Methods for Quantification of Short-Chain Fatty Acids in Human Fecal Samples by GC-MS.

The gut microbiota has attracted a great deal of interest in recent years due to its association with many diseases. Short-chain fatty acids (SCFAs), the end products of dietary fiber fermentation by the intestinal microbiota, are among the most frequently discussed gut metabolites. As the sample handling method greatly affects the integrity of data, this study investigated the most important parameters that affect the bias of SCFA comparisons in human fecal studies. An accurate gas chromatography-mass spectrometry (GC-MS) method was first established and validated for quantifying six SCFAs, including acetic, propionic, butyric, isobutyric, isovaleric, and valeric acids. To remove interfering species, we used butanol to extract SCFAs from acidified fecal suspensions. The validated quantification method was then applied to evaluate fecal sample handling protocols. We found that lyophilization of fecal samples can not only minimize bias due to the water content but also provide better stability of SCFAs. Six SCFAs were stable and that their recoveries were higher than 90% after lyophilization. Lyophilization of a large fecal sample is extremely time-consuming, and 1 g of fecal sample is suggested for lyophilization to minimize sampling bias. The interindividual difference was significantly higher than the intra-individual difference when using 1 g of fecal sample to study SCFAs. Finally, an effective protocol from sample collection to GC-MS analysis was proposed. As SCFAs have been shown to play an important role in health maintenance and disease development, the proposed protocol is anticipated to be applicable to clinical studies to delineate the biological functions of each SCFA.

Establishing a mucosal gut microbial community in vitro using an artificial simulator.

The Twin Simulator of the Human Intestinal Microbial Ecosystem (TWINSHIME®) was initially developed to study the luminal gut microbiota of the ascending (AC), transverse (TC), and descending (DC) colon regions. Given the unique composition and potential importance of the mucosal microbiota for human health, the TWINSHIME was recently adapted to simulate the mucosal microbiota as well as the luminal community. It has been previously demonstrated that the luminal community in the TWINSHIME reaches a steady state within two weeks post inoculation, and is able to differentiate into region specific communities. However, less is known regarding the mucosal community structure and dynamics. During the current study, the luminal and mucosal communities in each region of the TWINSHIME were evaluated over the course of six weeks. Based on 16S rRNA gene sequencing and short chain fatty acid analysis, it was determined that both the luminal and mucosal communities reached stability 10-20 days after inoculation, and remained stable until the end of the experiment. Bioinformatics analysis revealed the formation of unique community structures between the mucosal and luminal phases in all three colon regions, yet these communities were similar to the inoculum. Specific colonizers of the mucus mainly belonged to the Firmicutes phylum and included Lachnospiraceae (AC/TC/DC), Ruminococcaceae and Eubacteriaceae (AC), Lactobacillaceae (AC/TC), Clostridiaceae and Erysipelotrichaceae (TC/DC). In contrast, Bacteroidaceae were enriched in the gut lumen of all three colon regions. The unique profile of short chain fatty acid (SCFA) production further demonstrated system stability, but also proved to be an area of marked differences between the in vitro system and in vivo reports. Results of this study demonstrate that it is possible to replicate the community structure and composition of the gut microbiota in vitro. Through implementation of this system, the human gut microbiota can be studied in a dynamic and continuous fashion.

The impact of short-chain fatty acids on GLP-1 and PYY secretion from the isolated perfused rat colon.

The colonic epithelium harbors a large number of endocrine cells, but little is known about the endocrine functions of the colon. However, the high density of glucagon like peptide-1 (GLP-1)- and peptide-YY (PYY)-secreting L cells is of great interest because of the potential antidiabetic and antiobesity effects of GLP-1 and PYY. Short-chain fatty acids (SCFAs) produced by local bacterial fermentation are suggested to activate the colonic free fatty acid receptors FFAR2 (GPR43) and FFAR3 (GPR41), stimulating the colonic L cells. We used the isolated perfused rat colon as a model of colonic endocrine secretion and studied the effects of the predominant SCFAs formed: acetate, propionate, and butyrate. We show that luminal and especially vascular infusion of acetate and butyrate significantly increases colonic GLP-1 secretion, and to a minor extent also PYY secretion, but only after enhancement of intracellular cAMP. Propionate neither affected GLP-1 nor PYY secretion whether administered luminally or vascularly. A FFAR2- and FFAR3-specific agonist [( S)-2-(4-chlorophenyl)-3,3-dimethyl- N-(5-phenylthiazol-2-yl)butamide (CFMB)/ AR420626 ] had no effect on colonic GLP-1 output, and a FFAR3 antagonist ( AR399519 ) did not decrease the SCFA-induced GLP-1 response. However, the voltage-gated Ca2+-channel blocker nifedipine, the KATP-channel opener diazoxide, and the ATP synthesis inhibitor 2,4-dinitrophenol completely abolished the responses. FFAR2 receptor studies confirmed low-potent partial agonism of acetate, propionate, and butyrate, compared with CFMB, which is a full agonist with ~750-fold higher potency than the SCFAs. In conclusion, SCFAs may increase colonic GLP-1/PYY secretion, but FFAR2/FFAR3 do not seem to be involved. Rather, SCFAs are metabolized and appear to function as a colonocyte energy source. NEW & NOTEWORTHY By the use of in situ isolated perfused rat colon we show that short-chain fatty acids (SCFAs) primarily are used as a colonocyte energy source in the rat, subsequently triggering glucagon like peptide-1 (GLP-1) secretion independent of the free fatty acid receptors FFAR2 and FFAR3. Opposite many previous studies on SCFAs and FFAR2/FFAR3 and GLP-1 secretion, this experimental model allows investigation of the physiological interactions between luminal nutrients and secretion from cells whose function depend critically on their blood supply as well as nerve and paracrine interactions.

Impact of residual and therapeutic doses of ciprofloxacin in the human-flora-associated mice model.

A study was conducted to evaluate the effects of therapeutic and residual doses of ciprofloxacin on the human intestinal flora implanted into germ-free mice. Ciprofloxacin was administered daily via drinking water at concentrations to provide doses of 0, 0.125, 1.25, and 12.5mg/kg b.w. Changes in the intestinal flora composition, alteration in bacterial enzyme activities, fecal short chain fatty acid concentration and bacterial cellular fatty acid profiles, overgrowth of resistant bacteria, and disruption of the colonization barrier were the endpoints evaluated in the feces of human-flora-associated (HFA) mice. Ciprofloxacin at all tested doses decreased significantly the aerobic populations and particularly the population of Enterobacteriaceae. Selection of resistant Bacteroides fragilis group was noticed in HFA mice receiving 12.5mg/kg b.w. In mice challenged with a Salmonella strain, exogenous Salmonella persisted in the feces of all treated mice indicating that the flora responsible for the colonization barrier effect was disturbed by the antibiotic treatment. None of the studied metabolic parameters of the flora were affected by ciprofloxacin at any dose level. Under the experimental conditions of the study, the no-observed-effect level of ciprofloxacin was found to be less than 0.125 mg/kg b.w.

The effect of narasin on apparent nitrogen digestibility and large intestine volatile fatty acid concentrations in finishing swine.

The effect of narasin on apparent nitrogen and dry matter digestibilities and large intestine VFA concentrations in finishing swine was investigated. The study used 21 crossbred barrows averaging 72 kg. Seven blocks were formed on the basis of pretreatment dry matter digestibility, and barrows were randomly assigned to three treatments in each block. Treatments consisted of a control (C) and narasin (N15 and N30) applied at 15 and 30 ppm, respectively. Fecal and urine samples were collected. Upon the completion of the digestibility work, intestinal samples were taken from three locations, and VFA concentrations for each animal were measured. Weight gains for the N15 and N30 treatments were increased 3.0 and 6.0% (not significant), respectively, over control. Fecal nitrogen was decreased (P < .05) in the narasin-fed barrows, and apparent nitrogen digestibility was increased (P < .05). Neither nitrogen retention nor urinary nitrogen excretion was altered (P > .05) due to narasin. There were no increases (P > .05) in apparent dry matter digestibility due to narasin. Analysis of pooled colon samples showed an increase (P < .05) in the concentration of propionic acid in relation to acetic and butyric in the narasin-fed barrows. Butyric acid was reduced (P < .05) in the transverse colon of narasin-fed barrows. In summary, narasin administration to finishing barrows resulted in improved apparent nitrogen digestibility, thus decreasing fecal nitrogen, and increased relative concentrations of propionic acid in the large intestine.

Luminal short-chain fatty acids and postresection intestinal adaptation.

BACKGROUND: Short-chain fatty acids (SCFAs) reportedly have a trophic effect on the small intestine. However, it is unclear if this is a local or primarily systemic effect. Loss of the ileocolonic junction (ICJ) may result in increased SCFAs and bacteria in the small intestine from colonic reflux. Our aim was to evaluate the effect of bypass of the ICJ on intestinal SCFA content and postresection adaptation. METHODS: Thirty dogs were studied: transection control (TC, n = 10), distal resection of 50% intestine (DR, n = 10), and distal resection with bypass of ICJ (DRBP, n = 10). Animals were killed at 4 and 12 weeks. Luminal SCFAs and bacteria and adaptation of the small intestine were evaluated. RESULTS: Caloric intake was significantly less in the two resected groups (67 +/- 3 DR and 63 +/- 3, DRBP vs 78 +/- 5 kcal/kg/d TC, p < .05). Body weight and albumin levels were decreased at 12 weeks but were similar between the resected groups (81% +/- 3% and 74% +/- 6% initial and 1.9 +/- 0.1 and 2.1 +/- 0.2 g/dL, DR and DRBP, respectively). Steatorrhea was present for 12 weeks after resection and was greater after DRBP (14.2% +/- 3.8% vs 8.6% +/- 1.9% at 4 weeks and 13.6% +/- 2.5% vs 6.7% +/- 0.6% at 12 weeks, p < .05). Bypassed animals had elevated intraluminal SCFA content (3126 +/- 1094 vs 1791 +/- 538 DR and 1600 +/- 446 micrograms/mL TC, p < .05) and anaerobic bacterial counts (100% vs 50% and 44%, respectively). Tissue inflammation and myeloperoxidase activity were similar. Small intestinal length (174 +/- 10 and 180 +/- 10 cm) and circumference (5.2 +/- 0.4 and 5.2 +/- 0.3 cm) increased to a similar extent in both resected groups at 12 weeks. Thickness of mucosa (1939 +/- 162 vs 1662 +/- 162 microns) and muscle (865 +/- 45 vs 978 +/- 79 microns) layers were similar after DR and DRBP. CONCLUSION: (1) Bypass of the ICJ after distal resection results in increased growth of anaerobic bacteria and luminal SCFA and is associated with more marked steatorrhea. (2) Bypass of the ICJ does not influence structural adaptation of the small intestine. (3) These findings do not support a local trophic effect for SCFA.